The present invention relates to combined or composite enzyme systems for treating microbially produced extracellular polymers, present or which build up on surfaces of cooling water towers and in paper making broke water. Such extracellular polymers plus microbial cells are also known as biofilm or microbial slime.
Microbially produced extracellular polymers can build up, retard heat transfer and restrict water flow through cooling water systems. Controlling slime-forming bacteria by applying toxic chemicals is becoming increasingly unacceptable due to environmental problems. In addition, the efficacy of the toxicants is minimized by the slime itself, since the extracellular polysaccharide enveloping microorganisms are largely impenetrable.
Toxicants cannot adequately control large populations of attached bacteria and they are effective mainly against floating microorganisms. Although surfactants and dispersants which penetrate and help loosen slime can enhance the activity of toxicants, they are nonspecific and may have deleterious effects on the industrial process.
This invention describes use of enzymes which have the advantage of being both specific and non-toxic. The approach is designed to (a) enhance the removal of slime where it has formed, (b) prevent the build-up of slime, and (c) improve the efficacy of biocides against sessile bacteria. The enzymes specifically attack the slime layer surrounding the bacteria. Consequently, the microorganisms become planktonic--harmless in terms of biofilm production--and are rendered susceptible to biocides. The enzymes also act to maintain a clean surface (see FIG. 6 and remarks).
Examples of prior art single enzyme formulations are: those found in 3,773,623, Hatcher, Economics Laboratories, Inc., where the slime formulation in industrial water such as white water from pulp and paper mills is retarded by controlling amounts of enzyme levan hydrolase.
Also, 4,055,467, Christensen (Nalco) describes a slime and an industrial process whereby slime can be dispersed and prevented by treating said slime with a few ppm of the enzyme, Rhozyme HP-150, a pentosanase-hexosanase and 3,824,184, Hatcher (Economics Laboratories, Inc.) describes a slime formation controlled by intentionally adding to industiral water the controlled amounts of enzyme levan hydrolase.
Additionally, 4,684,469, Pedersen et al. (Accolab, Inc.) discloses a method of a two-component biocidal composition suitable for controlling slime. The preparation consists of a biocide and a polysaccharide degrading enzyme.
As to the biocides, generally methylene-bis-thiocyanate has been preferred. Other operable biocides include chlorophenate compounds, such as pentachlorophenates and trichlorophenates; organomercurial compounds, such as phenylmercuric acid; carbamate compounds, such as methyldithiocarbamates, ethylenebisdithiocarbamates, and dimethyldithiocarbamates; carbonate compounds such as cyanodithioimidocarbonates; thiocyanates such as chloroethylene thiocyanate compounds; and other biocides such as bromo-hydroxyacetophenone compounds, benzothiazole compounds, ethylene diamine compounds, nitrilopropionamides, bromopropionamides, bromo-acetoxybutenes, bromopropanolaldehyde compounds, bis-trichloromethyl sulfones, bimethyl hydantoin compounds, and the like mixtures of biocides can also be used.
The biocide methylene-bis-thiocyanate has proven to be particularly effective in the context of this invention, as has a combination of dimethyldithiocarbamate and disodium ethylenebisdithiocarbamate.
The advantages of the enzyme blend composition over the use of biocides to control bacteria are that the biocides constitute toxicants in the system and pollution problems are ever present. The advantage of the present formulation over the formulation of a single enzyme plus biocide is that the single enzyme attacks only one narrow band of carbohydrate polymers whereas the present invention improves the range of attack by combining activities of a cellulase and an alpha-amylase along with the basic protease, broadly attacking the carbohydrate polymer and protein surrounding the bacteria. A specific formulation embodying ratios, for the present use of multiple enzyme preparations, is 2 parts cellulase, 1 part alpha-amulase, and 1 part protease. In this formulation, the alpha-amylase is at least 1 and can be slightly over 1 part. The protease which is set at 1 may actually be 0.5 to 1 part, and the cellulase is set at 2 parts.
A preferred composition is 2 parts cellulase, 1 part alpha-amylase and 1 part protease. In the composition cerulase may be substituted for cellulase.
In general, most enzymes are used in a dosage of 2 to 100 ppm and many are from 2 to 10 parts per million. The enzymes can be obtained from many chemical suppliers such as American Cyanamid, Betz, Beckman, Dearborn Chemical, Economics Laboratory, Inc., Merck, Nalco, Vineland Chemical, and the like.
The concentration of enzyme required for effectiveness in this invention varies greatly and can depend upon the conditions such as temperature and pH of the water, the microbial count and the type of industrial water being treated. The lower and upper limits of the required concentrations will substantially depend upon the specific enzyme or combination of enzymes used. For example, a highly effective enzyme can require a concentration of mainly about 1 or 2 parts enzyme to one million parts industrial water in the context of this invention, while another enzyme may require a minimum concentration of 80 to 100 ppm.
In contrast to the prior art, this formulation is both more specific and non-toxic. In view of this invention and in comparison with the prior art, it can be said that the present composition has the same over target polymers but digests them more efficiently because of the combined enzyme activities of alpha-amylase, cellulase, and the protease. Moreover, the cellulase is a unique enzyme component which allows this efficiency to take place. The alpha-amylase and the protease nick the microbial slime and allow the cellulase access to digest the slime exopolymer more effectively.
It is noted as a matter of general mechanisms, that the alpha-amylase alone does not give slime protection or remove slime. It attacks the alpha-linkage between glucose molecules. It nicks the outside of the slime molecule, so that the cellulase can enter and attack said carbohydrate molecule. The protease attacks extracellular protein molecules.
Up to this time, enzyme treatment of industrial slime or slime polymer made by bacteria consisted of a single enzyme, for example levanase. Levanase would break down a polymer of levan into its subunits (fructose). However, after the levanase would be used on the slime levan, resistant bacteria would still remain to proliferate. Further applications of levanase were ineffective because the polymer it attacks was no longer present. The levan polymer would be gone, but other slime polymers would still be there and the bacteria would flourish. Although other enzyme preparations have been used in the marketplace, for example EDC, a levan hydrolyzer (Sunoco), there has been no combination of enzymes that would actually attack polymer made by Pseudomonas bacteria and other bacteria in the field, such as Klebsiella, Acinetobacter, Flavobacterium, Enterobacter, and Aerobacter, which were rich in glucose, mannose and gulose sugars arranged in polymers.
Now, in a generalized process and in response to the prior art above, the present invention has taken a clear culture of Pseudomonas bacteria and made them produce a slime polymer in a low substrate environment. Second, the invention has taken a composite of microorganisms from the field blended with and grown together both at the laboratory and under field conditions, simulated cooling tower water and utility water.
The results indicate that the maximum removal of carbohydrate layers from pending bacteria has occurred. Thus utilizing a new blend of enzymes has a superior result, especially if the enzyme utilization was found to be useful in the very prevalent Pseudomonas bacteria.
A variety of enzymes were utilized in testing against Pseudomonas bacteria. From 42 preparations of enzymes, three types of enzymes were found to be effective on slime produced by Pseudomonas bacteria. First, alpha-amylase was found to attack bacterial slime. Second, protease has been found also to have an effect on bacterial slime. Then it was found that a combination enzyme treatment with amylase, cellulase, and protease was effective in removing the biofilm. Neither one by themselves, however, would remove enough slime to be effective. The blend of cellulase, amylase and protease was the answer.